Bioactivation of cinnamic alcohol in a reconstructed human epidermis model and evaluation of sensitizing potency of the identified metabolites

Background Cinnamic alcohol is a natural compound, widely used in fragrances, which can cause allergic contact dermatitis. Cinnamic alcohol lacks intrinsic reactivity and autoxidation or metabolic activation is necessary for it to act as a sensitizer. Methods Bioactivation of cinnamic alcohol was explored using human liver microsomes, human liver S9 and SkinEthic™ Reconstructed Human Epidermis. A targeted multiple reaction monitoring mass spectrometry method was employed to study and quantify cinnamic alcohol along with eight potential phase I or phase II metabolites. The reconstructed human epidermis model, treated with cinnamic alcohol, was also analyzed with a non-targeted high-resolution mass spectrometry method to identify metabolites not included in the targeted method. Results Two metabolites identified with the targeted method, namely, pOH-cinnamic alcohol and pOH-cinnamic aldehyde, have not previously been identified in a metabolic in vitro system. Their reactivity toward biologically relevant nucleophiles was investigated and compared to their sensitizing potency in vivo in the murine local lymph node assay (LLNA). According to the LLNA, the pOH-cinnamic alcohol is non-sensitizing and pOH-cinnamic aldehyde is a moderate sensitizer. This makes pOH-cinnamic aldehyde less sensitizing than cinnamic aldehyde, which has been found to be a strong sensitizer in the LLNA. This difference in sensitizing potency was supported by the reactivity experiments. Cinnamic sulfate, previously proposed as a potential reactive metabolite of cinnamic alcohol, was not detected in any of the incubations. In addition, experiments examining the reactivity of cinnamic sulfate toward a model peptide revealed no evidence of adduct formation. The only additional metabolite that could be identified with the non-targeted method was a dioxolan derivative. Whether or not this metabolite, or one of its precursors, could contribute to the sensitizing potency of cinnamic alcohol would need further investigation. Discussion Cinnamic alcohol is one of the most common fragrance allergens and as it is more effective to patch test with the actual sensitizer than with the prohapten itself, it is important to identify metabolites with sensitizing potency. Further, improved knowledge of metabolic transformations occurring in the skin can improve prediction models for safety assessment of skin products.


TABLE OF CONTENTS TARGETED LC-MS/MS ANALYSIS OF FORMED METABOLITES IN HUMAN LIVER MICROSOMES AND SKINETHIC
. Accuracy, expressed as % relative error (%RE), and the precision of the method, expressed as % relative standard deviation (%RSD), calculated after analysis of cinnamic alcohol at three different concentration levels. .Table S3.Accuracy, expressed as % relative error (%RE), and the precision of the method, expressed as % relative standard deviation (%RSD), calculated after analysis of cinnamic alcohol at three diIerent concentration levels.Non-targeted LC-MS/MS analysis of formed metabolites in SkinEthic TM reconstructed human epidermis

Sample
b Fragmentation structures were predicted with CFM-ID 4.0 1 (available at https://cfmid.wishartlab.com/).b Fragmentation structures were predicted with CFM-ID 4.0 1 (available at https://cfmid.wishartlab.com/).a Groups of mice were treated with test substance in 5 different concentrations, on the dorsum of both ears for 3 consecutive days.Sham treated control animals received vehicle alone.On day 5, all mice were injected intravenously with PBS (250 µl) containing 20 µCi of [ 3 H]-methylthymidine.After 5 h the mice were sacrificed, the draining lymph nodes were excised and pooled for each group, single cell suspensions of lymph-node cells were prepared, and the thymidine incorporation into DNA was measured by β-scintillation counting.The increase in thymidine incorporation relative to vehicle-treated controls was derived for each experimental group and recorded as stimulation index 3 .The EC3 values (the estimated concentration required to induce an SI of 3) were calculated using linear interpolation.The sensitizing potency was classified to the following: <0.1%, extreme; ≥0.1 to <1%, strong; ≥1 to <10%, moderate; and ≥10%, weak 4 .b Molarity was calculated based on the molecular weight of cinnamic alcohol.
Figure S1.Compound name, parent ion and fragments used in the MRM method for the quantification of the formed metabolites.

Figure S2 .
Figure S2.Calibration curves and the equivalent residuals plot for each compound quantified.

Figure S5 .
Figure S5.Data could suggest the presence of a dioxolan hydroperoxide in RHE incubated with cinnamic alcohol.This is supported by: A) At RT 10.75 min, the full MS spectrum indicates: m/z 315.1226 and m/z 337.1046.These measured m/z values show a mass deviation of less than 2 ppm from the theoretical ions corresponding to [M+H] + and [M+Na] + , respectively; B) The MS/MS spectrum of m/z 315.1226 shows the presence of m/z 133.06, 121.06, 105.07, and 91.05, indicating that it is a derivative of cinnamic alcohol (Figure S1).

Figure S6 .
Figure S6.Data could suggest the presence of a dioxolan aldehyde in RHE incubated with cinnamic alcohol.This is supported by: A) An extracted ion chromatogram of m/z 281.1177 (5 ppm mass tolerance) in RHE medium incubated with cinnamic alcohol for 24 h revealed an increase in the area of several peaks between RT 6.6-12.0min over time (2h to 24h); B) Two of the formed peaks recorded the accurate MS/MS of m/z 281.1169 at retention time 8.44 (upper spectrum) and 9.38 min (lower spectrum), respectively, possibly belonging to the dioxolan cinnamic aldehyde.Adding the theoretical structure to an in silico fragmentation software (CFM-ID 4.0 1 , available at https://cfmid.wishartlab.com/)revealed several similar fragments are observed such as m/z 281.1172 [M+H] + , 263.1066, 251.1066, 175.0753, 131.0491 and 91.0542.

Figure S7 .
Figure S7.Data suggesting the presence of an epoxy cinnamic alcohol-glutathione adduct A) Proposed structure and mechanism, adapted from Charpentier et al., for the formation of a glutathione adduct with epoxy cinnamic alcohol 2 .The adduct is formed by an SN2 reaction of glutathione to epoxy cinnamic alcohol.This formation of this adduct is supported by: B) The full MS (RT = 1.11 min) with an addition of C9H10N3O2 to glutathione corresponds to a mass accuracy of -2.0 ppm.C) The acquired MS/MS spectrum of m/z 458.1588 shows the characteristic fragmentation of glutathione indicated in the figure by asterisks (*), and possible losses are detailed.

Figure S8 .
Figure S8.Data suggesting the presence of a cinnamic aldehyde-glutathione adduct A) Proposed structure and mechanism, adapted from Charpentier et al., for the formation of a glutathione adduct with cinnamic aldehyde 2 .The first step is a Michael addition of glutathione to cinnamic aldehyde followed by a reduction of the aldehyde to an alcohol.This formation of this adduct is supported by: B) The full MS (RT = 2.15 min) with an addition of C10H10O to glutathione corresponds to a mass accuracy of -1.1 ppm.C) The acquired MS/MS spectrum of m/z 442.1642 shows the characteristic fragmentation of glutathione, indicated in the figure by asterisks (*), and possible losses are detailed.

Table S4 .
Suggested fragmentation structures for the dioxolan derivate identified in the non-target screening.

Table S1 .
Limit of detection (LoD) and limit of quantification (LoQ) for all the metabolites expressed in nM, pg/mL and pg on column.

Table S2 .
Linearity assessment including range in µM, R squared, and slope values for the calibration curves of each analyte.

Table S4 .
Suggested fragmentation structures for the dioxolan derivate identified in the non-target screening.b b Fragmentation structures were predicted with CFM-ID 4.0 1 (available at https://cfmid.wishartlab.com/).

Table S5 .
Suggested fragmentation structures for the epoxy cinnamic alcohol-glutathione adduct identified in the non-target screening.b

Table S6 .
Suggested fragmentation structures for the glutathione-cinnamic aldehyde adduct identified in the non-target screening.b

Table S7 .
Results from the murine local lymph node assay (LLNA) a : Sensitization experiments of the two pOH-cinnamic compounds and the control cinnamic aldehyde.